In various fields including the fields of pharmaceuticals and foods, there has been an increasing need for a technique for separating an optical isomer to thereby collect the one useful optical isomer. For example, research has been conducted on a process for producing drugs and perfumes by using an asymmetric hydrogenation (reducing) enzyme having a function of selectively oxidizing/reducing or deracemizing one of optical isomers.
A chemical change in vivo is usually catalyzed by an enzyme. A bioreactor is an example of one of the systems of utilizing such an action or mechanism of these enzymes in the production or analysis of useful substances.
An immobilized enzyme, which has been bonded to an insoluble carrier, plays a central role in a bioreactor. By use of the immobilized enzyme, a product can easily be separated from the enzyme serving as a catalyst. The immobilized enzyme has widely been used in the fields of research, medical care, analysis and industry. The central part of the bioreactor causing a chemical reaction is a reaction element, and a purified enzyme, organelle or cell per se is used for the purpose of converting a raw material into a product, or analyzing by utilizing a chemical change. Since the reaction element must remain in a reactor and be able to be used repeatedly, the reaction element is immobilized by various methods (with respect to details of utilization of these enzymes, for example, it is possible to refer to Non-Patent Document 1).
However, since the enzyme has numerous problems such as requiring extensive costs for purification and the purified enzyme often being unstable, there is still room for improvement. For this reason, there is an example wherein a microorganism containing the target enzyme was immobilized as is instead of the purified enzyme. Examples thereof may include an example wherein microorganisms containing aspartase were immobilized to produce L-aspartic acid, and an example wherein L-alanine was continuously produced by using aspartic acid able to be produced in this plant as a raw material (see, for example, Non-Patent Document 1 and Non-Patent Document 2).
As indicated in Non-Patent Document 3, with respect to a redox catalyst seen from an industrial point of view, it is considered that a method of utilizing functions per se of dehydrogenase and coenzyme, which are present in cells, such as microorganisms, yeasts and cultured plant cells, is superior in terms of cost, and that the excess cost burden required to isolate and purify oxidoreductase and coenzyme from boint is not worth the cost of an operation of stabilizing an enzyme, and conjugating a reaction (ketone→alcohol) with a reaction (coenzyme NADH→NAD+) or a reverse reaction thereof.
A medium-chain dehydrogenase/reductase (MDR) type of alcohol dehydrogenase (ADS) is able to catalyze an oxidation reaction of a secondary alcohol using coenzyme NAD(H), and according to the review of Non-Patent Document 4, catalytic zinc, which acts as an electron transfer system, and structural zinc, which acts to maintain structure, are both present in this dehydrogenase, and regularity is observed in the amino acid sequence thereof at the locations of amino acids such as cysteine and histidine.
Since commercially available alcohol dehydrogenation (oxidation)/hydrogenation(reduction) enzymes are dependent on electron transfer between coenzymes produced by these microorganisms (NADH→NAD+) and the intrinsic zinc, a reaction occurs by addition of coenzyme. An example of an enzyme product obtained by heat resistance treatment of a secondary alcohol dehydrogenase may be Secondary Alcohol Dehydrogenase A available from Thermostable Enzyme Laboratory Co., Ltd.
A process in which an asymmetric hydrogenase is used as is without being purified by a microorganism can be summarized as follows: 1) searching for and collecting a microorganism that induces a substrate transformation reaction, 2) culturing (for six months), and 3) carrying out a microbial reaction. Examples of problems encountered in this case may include: 1) burden in terms of time and costs required to find the microorganism and develop for practical application, and 2) requirement of the supervision of a specialist familiar with microbial procedures. Examples of problems associated with asymmetric hydrogenation (reduction) reactions using an organic metal catalyst (such as BINAP), for which there are many as 500,000 to 1,000,000 types, may include: 1) cost burden attributable to the reaction system in which hydrogen is pressurized in an organic solvent, 2) residual harmful metal (such as ruthenium) following the reaction, and 3) low stereoselectivity (85% ee to 95% ee in nearly all cases).
The merit of an asymmetric dehydrogenation (oxidation) catalyst is that it can play an auxiliary role in this conventional asymmetric technology. For example, an asymmetric dehydrogenation catalyst can be used for the purpose of selectively asymmetrically oxidizing and removing an unnecessary enantiomer produced with BINAP. An asymmetric oxidation catalyst in this case may preferably use a reaction system that 1) does not require the supervision of a specialist, 2) uses an aqueous medium, and 3) proceeds with low cost due to high stereoselectivity (100% ee).
Dehydrogenases involved in redox reactions have been reported that have iron, which has a broader range of electron mobility than zinc. For example, Non-Patent Document 5 reports a primary alcohol-specific dehydrogenase, specific for methanol or ethanol and the like, that has a quinoprotein as a coenzyme (which may also be referred to as “CC1”). A general reaction formula of this PQQ-dehydrogenase is as indicated below, and oxygen supply is involved in the reaction.4Fe2+—CC1+8Hin++O2→4Fe3+—CC1+2H2O+4Hout+  [Chemical Formula 1]
However, Non-Patent Document 5 does not report an asymmetric dehydrogenation reaction of a secondary alcohol in the presence of PQQ.
In addition, according to Non-Patent Document 6, a cytochrome oxidase (which may also be referred to as “CC2”) is reported in which the electron transfer of iron is involved.2Fe2+—CC2+ethanol⇄2Fe3+-reduced-CC2 acetoaldehyde  [Chemical Formula 2]
Although an enzyme is not involved in this reaction, there is no report in Non-Patent Document 6 of an asymmetric dehydrogenation reaction of a secondary alcohol in Non-Patent Document 6 in the same manner as PQQ.
According to Non-Patent Document 7, a peroxidase is reported in which the electron transfer of iron is involved (which may also be referred to as “CC3”).H2O2+CC3—H2→2H2O+CC3  [Chemical Formula 3]
An enzyme is also not involved in this reaction, and there is no report of an asymmetric dehydrogenation reaction of a secondary alcohol in Non-Patent Document 7 as well in the same manner as PQQ and cytochrome oxidase. The case of catalase, in which the electron transfer of iron is involved (which may also be referred to as “CC4”), is similar in that an enzyme is not involved.CC4+2H2O2→CC4+O2+2H2O  [Chemical Formula 4]
In this manner, there are no known reports relating to an asymmetric dehydrogenase of a secondary alcohol based on the electron transfer of iron.
The invention of a novel asymmetric dehydrogenation (oxidation) catalyst that demonstrates an asymmetric dehydrogenation reaction of a secondary alcohol and is dependent on iron electron transfer and oxygen would make it possible to selectively asymmetrically oxidize and remove unnecessary enantiomers produced with BINAP. Moreover, since coenzyme NAD(P) is not present, it would be possible to realize a reaction system in which 1) the need for the supervision of a specialist is eliminated, while only requiring oxygen, 2) an aqueous medium is used, and 3) the reaction proceeds at low cost due to high stereoselectivity (100% ee).
Cytochrome P450 monooxygenase (P450) is further attracting attention with respect dependence on an iron electron transfer system and oxygen (O2) and, dependence on a coenzyme (NAD(P) and/or FAD) depending on the case. According to Non-Patent Document 14, reduced iron was indicated to act on xenobiotic metabolism and the biosynthesis of secondary metabolites such as steroids, fatty acids, terpenoids or flovonoids by using oxygen atoms as oxidizing agent and catalyzing stereoselective and regioselective hydroxylation, epoxidation, dehalogenation and other oxidation reactions.
According to Non-Patent Document 15, P450 is formed for the purpose of acting by selecting different in molecules in all living organisms, and roughly 20 types have been identified in bacteria, roughly 60 types in animals, and several hundred types in plants. One reason of this large number of types in plants is that it is the result of having been avariciously acquired to a greater degree than in other living organisms as a result of developing, evolving and adapting in a specific environment as indicated in Non-Patent Document 16.
According to Non-Patent Document 17, a hemophore is an iron-capturing protein intrinsic to iron-binding protein, and is described as being widely present in pyrroloquinoline quinone-alcohol dehydrogenase (PQQ-ADD), cytochrome oxidase and P450 localized in membrane protein, and in peroxidase, catalase and ABC transporter.
Patent Document 1 indicates an example wherein an optically active alcohol was resolved at an optical purity of about 100% ee from a crude protein derived from animals and plants, and an optically active alcohol (100% ee, yield: 50%) is synthesized by combining a first step for extracting a water-soluble protein from grains or beans, a second step for encapsulating the protein in a calcium alginate gel, and a third step for carrying out an asymmetric oxidation conversion reaction of a substrate using the encapsulated protein as a catalyst. In addition, in Patent Document 2, an optically active alcohol (100% ee) is synthesized by combining a first step for extracting a water-soluble protein selected from egg white and ovalbumin separated from egg white, a second step for encapsulating the protein in calcium alginate, and a third step of carrying out an asymmetric oxidation conversion reaction of a substrate using the encapsulated protein as a catalyst.
Moreover, Patent Document 3 discloses a production process wherein, after having encapsulated a water-soluble protein from grains or beans in in calcium alginate gel and oxidizing in air, a protein fraction eluted in warm water is precipitated with ammonium sulfate, chemically modified with glutaraldehyde and formed into a powder, and the resolution/synthesis of an optically active alcohol using that process.
In addition, according to Non-Patent Document 8, a PEG-coated lipase complex (white powder) is obtained by coating lipase with polyethylene glycol (PEG) and forming an emulsion by adding toluene followed by subjecting to freeze-drying treatment for 24 hours. Moreover, an asymmetric acylation reaction of alcohol in an ionic liquid ([Bmin][PF6]) has been studied, and a dramatic improvement in catalyst activity was reported to be observed as a result of coating with PEG (IL1-PS: Non-Patent Document 13).
The size of the lipase hydrolase market in the chemical industry is considered to be small in comparison with the asymmetric hydrogenation market relating to BINAP catalyst and microbial hydrogenation, being only 1/100 to 1/1000 the size of that market, while the size of the asymmetric redox market is large. Thus, a technology for coating PEG onto a certain asymmetric oxidase instead of lipase would be deeply interesting in terms of the market. This PEG-coated oxidase would be able to function as a cocatalyst that may preferably remove unnecessary enantiomers formed in BINAP reactions. However, existing PEG coating treatment requires the consumption of a large amount of toluene, and has concerns over economic and environmental burdens. Therefore, there is a strong desire for a coating technology that does not use an organic solvent (such as toluene) that results in such concerns over economic and environmental burdens.
Typical examples of methods used to produce an immobilized enzyme may include:
(1) a carrier binding method wherein the extracted and purified enzyme is bound to a water-insoluble carrier such as a derivative of a polysaccharides such as cellulose, dextran or agarose or a polyacrylamide gel;
(2) a chemical modification method wherein the extracted and purified enzyme is immobilized by forming a chemically modified bond between the extracted and purified enzymes using a reagent having two or more functional groups; and
(3) a (microcapsule type) encapsulating method wherein the extracted and purified enzyme is incorporated in a fine matrix of a gel such as alginate, starch, konjak (devil's tongue jelly), polyacrylamide gel or polyvinyl alcohol, or coated with a semitransparent film.
In general, the significance of these immobilized enzymes is that they may be preferably recovered (and more preferably, recovered and then reused) after reacting. Although treatment involving encapsulation in calcium alginate gel followed by oxidizing in air as described in Patent Document 3 is an example of a novel technology that further advances this immobilization technology by oxidizing an immobilized biomaterial in air to transform to a beneficial material, definitive evidence has yet to be observed regarding the estimated mechanism of this treatment with respect to the formation of a disulfide bond and/or reduction of iron molecules in a hemophore (iron-capturing protein) accompanying air oxidation of cysteine within a protein, and the change of the PC per se to water solubility resulting in a “exudation” effect simultaneous to an “asymmetric oxidation” effect.